The invention uses a carrier medium to carry different reagents for signal generation and signal localization in the assay. The carrier medium comprises:                (i) Solvent (e.g., aqueous solution including buffer, salt solution, water; organic solvent, e.g., alcohols such as ethanol, propanol; ethers such as tetrahydrofuran (THF), and polar aprotic solvents such as dimethyl sulfoxide (DMSO);        (ii) Thickener, which dissolves in the solvent as a colloid mixture that forms an internal structure giving the resulting carrier medium properties ranging from those of a high viscosity fluid to a gel. Gel type carrier media have the appearance of a solid while being mostly composed of the solvent. Examples include polymers such as polyacrylamide and polyisobutylene; polysaccharides such as starches, cellulose, alginates obtained from brown algae, agar, carrageenan, pectin; natural gums such as locust bean gum and guar gum; proteins such as collagen, albumin and gelatin.        
In some embodiments, the carrier medium may also include a signal developing reagent which is a material that converts signal precursor molecules to a state in which they generate a measurable signal. In other embodiments, a signal developing reagent is not necessary because the plurality of signal precursor molecules is converted to a plurality of detectable signal molecules by physical means such as change in temperature, change of pH, sonication, light irradiation or microwave heating.
The functions of the carrier medium are:                (i) To hinder the diffusion of the signal molecules, leading to signal accumulation.        (ii) To improve the readability (sharpness, prolongation of signal retention time) of the signal on the solid phase platform, and thus enhance the sensitivity.        
In some embodiments, where the conversion of the plurality of signal precursor molecules to a plurality of detectable signal generating molecules is brought about by chemical or biochemical means, the carrier medium has a third function:                (iii) To generate a signal by converting a plurality of signal precursor molecules to a plurality of detectable signal generating molecules.        
For assays using detection of visible light, the carrier medium is substantially optically transparent.
Many types of target-receptor assays have been used to detect the presence of various target substances in body fluids such as urine, blood, serum, plasma, saliva or extraction solutions of faeces. These assays typically involve antigen antibody reactions, synthetic conjugates with radioactive, enzymatic, fluorescent, luminescent, chemiluminescent, or visually observable metal tags, and use specially designed reaction chambers. In all such assays, there is a receptor, e.g., an antibody, which is specific for the selected target, e.g., antigen, and a means for detecting the presence, and often the amount, of the target-receptor reaction product. Many current tests are designed to provide a semi-quantitative or quantitative determination but, in many circumstances, all that is required is a qualitative detection providing a positive or negative indication of the presence of the target species. Examples of such qualitative assays include blood typing, most types of urinalysis and the very important faecal occult blood test as a screening assay for colorectal carcinoma. For these tests, visually observable indicia such as the accumulation of coloured particles, e.g., gold particles, the presence of agglutination or a colour change are preferred.
Nevertheless, qualitative assays must be very sensitive because of the often small concentration of the target of interest in the test fluid. Sandwich assays and other sensitive detection methods which use metal sols or other types of coloured particles have been developed. However, these techniques have not solved all of the problems encountered in rapid detection methods and further improvements are constantly being sought.
By way of example, in lateral flow sandwich assays, colloidal gold is often used as a label of a first antibody (1) whereas the other antibody (2) is fixed in a well-defined detection site on a membrane such as a nitrocellulose membrane. If the analyte in question is present in a sample, then the analyte reacts with the gold labelled antibody (1) and migrates to the nitrocellulose membrane-bound antibody (2). There it forms a sandwich and these sandwich complexes are then collected and concentrated in the detection site. This zone can be made more visible to a certain extent (amplified) by additional reaction with silver ions.
However, the analytical sensitivity is not outstanding and this technology is not readily applicable to certain assays, e.g., for the determination of thyroid-stimulating hormone (TSH), prostate specific antigen (PSA), Troponin I or Troponin T in the low—but diagnostically very important—concentration range.
Therefore, in order to make such assays more effective, other labels are used (named “signal amplification precursor molecules”) which can be amplified at the end of the determination reaction, for example at the end of the formation of the sandwich: antibody (2)fixed-analyte-{antibody (1)-label}.
If the amplification label is a microcapsule containing crystalline fluorescein diacetate (FDA)—consisting of millions of FDA molecules—then the amplification is effected after the determination reaction by disintegrating the microcapsule and hydrolysing the non-fluorescing FDA-molecules to fluorescing fluorescein molecules. This amplification is well proven and described in granted European patent number EP 1309867, the disclosure of which is incorporated herein by reference.
Normally the amplification reaction takes place in a solution of the releasing reagent. This leads to a slight decrease in analytical sensitivity from the ideal because of the dilution factor—the released fluorescein molecules become diluted in the reaction volume of the releasing reagent.
Unfortunately, in certain circumstances, the benefit of amplification can be offset or even outweighed by the disadvantage of signal dissipation. For example, if the detection or determination is performed on a membrane, e.g., in a lateral flow test strip, the addition of a solution of releasing reagent for enabling signal amplification results in diffusion of the amplified signal along the membrane. The released/amplified molecules are not localized and hence the signal may be difficult to detect even after amplification. The addition of any solution—after the underlying test reaction has been completed—leads to an enlargement of the detection line, spot or zone. Diffusion broadens the detection site and it is not possible to measure reliably the colour intensity of the detection site.
The prior art also includes examples where the label may be an enzyme with a high turnover number which, when it reacts with its substrate, forms very many reaction product molecules. Again, this is carried out in aqueous solution, more specifically in a buffer solution with the specific substrate molecules for the enzyme. One disadvantage of enzyme-based systems is that, because they are catalytic, the amplification starts when the conversion of the substrate occurs and this is an ongoing process. The amplification is dependent on the substrate concentration and on the time chosen for the enzymatic reaction. If no stopping reagent is added, the enzyme will work during the measurement time (and afterwards), so there is not a constant signal. Addition of a stopping agent will increase the dilution effect.